Summary: Poxviruses, and in particular vaccinia virus, have been utilized as systems for the expression of proteins in eukaryotic cells and as vectors for antigen delivery. Their ability to incorporate large amounts of DNA and wide host range leads to the expression and correct processing of a great variety of proteins in many cell lines. High level expression vectors have been constructed by expressing bacteriophage RNA polymerase genes in vaccinia. These vectors express high levels of any gene located behind the bacteriophage promoter. We have developed a series of vectors for generation of viral recombinants or conditional expression of target genes. Poxvirus vectors are customarily constructed by introducing foreign DNA into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been developed by our lab to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We have constructed and characterized direct ligation vectors engineered to contain restriction sites to fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters at the beginning of the thymidine kinase gene to utilize drug selection in the isolation of recombinants. These viruses provide a set of universally applicable, direct ligation, poxvirus cloning vectors, which extend the utility of poxvirus vectors for construction and expression of complex libraries. Our lab is currently working on the development of new poxvirus based vectors, including the development of direct ligation vectors for attenuated viruses with host range defects. These vectors will exhibit increased safety, especially for immunocompromised patients, since the infections will be self-limiting. One host-range, restricted-attenuated virus under consideration as a vector is modified virus ankara (MVA). We are also pursuing the development of chimeric vectors containing vaccinia virus DNA and bacterial origins of DNBA replication for the construction of host-range attenuated recombinants. In addition, the laboratory is evaluating the critical elements in the immune response generated by the present and potential attenuated vaccines against smallpox. The approaches include the elucidation of the viral proteins that elicit a humoral response, the evaluation of the heterogeneity in the vaccine and its influence on immunogenicity and the derivation of new assays to measure the critical components in the immune response to a smallpox vaccine.